Fourier Transform Infrared Spectroscopic Imaging-Derived Collagen Content and Maturity Correlates with Stress in the Aortic Wall of Abdominal Aortic Aneurysm Patients.

Abdominal aortic aneurysm (AAA) is a degenerative disease of the aorta characterized by severe disruption of the structural integrity of the aortic wall and its major molecular constituents. From the early stages of disease, elastin in the aorta becomes highly degraded and is replaced by collagen. Questions persist as to the contribution of collagen content, quality and maturity to the potential for rupture. Here, using our recently developed Fourier transform infrared imaging spectroscopy (FT-IRIS) method, we quantified collagen content and maturity in the wall of AAA tissues in pairs of specimens with different wall stresses. CT scans of AAAs from 12 patients were used to create finite element models to estimate stress in different regions of tissue. Each patient underwent elective repair of the AAA, and two segments of the AAA tissues from anatomic regions more proximal or distal with different wall stresses were evaluated by histology and FT-IRIS after excision. For each patient, collagen content was generally greater in the tissue location with lower wall stress, which corresponded to the more distal anatomic regions. The wall stress/collagen ratio was greater in the higher stress region compared to the lower stress region (1.01 ± 1.09 vs. 0.55 ± 0.084, p = 0.02). The higher stress region also corresponded to the location with reduced intraluminal thrombus thickness. Further, collagen maturity tended to decrease with increased collagen content (p = 0.068, R = 0.38). Together, these results suggest that an increase in less mature collagen content in AAA patients does not effectively compensate for the loss of elastin in the aortic wall, and results in a reduced capability to endure wall stresses.

Distinct macrophage phenotype and collagen organization within the intraluminal thrombus of abdominal aortic aneurysm.

OBJECTIVE: Little is known about the etiologic factors that lead to the occurrence of intraluminal thrombus (ILT) during abdominal aortic aneurysm (AAA) development. Recent work has suggested that macrophages may play an important role in progression of a number of other vascular diseases, including atherosclerosis; however, whether these cells are present within the ILT of a progressing AAA is unknown. The purpose of this work was to define the presence, phenotype, and spatial distribution of macrophages within the ILT excised from six patients. We hypothesized that the ILT contains a population of activated macrophages with a distinct, nonclassical phenotypic profile. METHODS: ILT samples were examined using histologic staining and immunofluorescent labeling for multiple markers of activated macrophages (cluster of differentiation [CD]45, CD68, human leukocyte antigen-DR, matrix metalloproteinase 9) and the additional markers α-smooth muscle actin, CD34, CD105, fetal liver kinase-1, and collagen I and III. RESULTS: Histologic staining revealed a distinct laminar organization of collagen within the shoulder region of the ILT lumen and a spatially heterogeneous cell composition within the ILT. Most of the cellular constituents of the ILT were in the luminal region and predominantly expressed markers of activated macrophages but also concurrently expressed α-smooth muscle actin, CD105, and synthesized collagen I and III. CONCLUSIONS: This report presents evidence for the presence of a distinct macrophage population within the luminal region of AAA ILT. These cells express a set of markers indicative of a unique population of activated macrophages. The exact contributions of these previously unrecognized cells to ILT formation and AAA pathobiology remains unknown.

Fourier transform infrared spectroscopy to quantify collagen and elastin in an in vitro model of extracellular matrix degradation in aorta.

Extracellular matrix (ECM) is a key component and regulator of many biological tissues including aorta. Several aortic pathologies are associated with significant changes in the composition of the matrix, especially in the content, quality and type of aortic structural proteins, collagen and elastin. The purpose of this study was to develop an infrared spectroscopic methodology that is comparable to biochemical assays to quantify collagen and elastin in aorta. Enzymatically degraded porcine aorta samples were used as a model of ECM degradation in abdominal aortic aneurysm (AAA). After enzymatic treatment, Fourier transform infrared (FTIR) spectra of the aortic tissue were acquired by an infrared fiber optic probe (IFOP) and FTIR imaging spectroscopy (FT-IRIS). Collagen and elastin content were quantified biochemically and partial least squares (PLS) models were developed to predict collagen and elastin content in aorta based on FTIR spectra. PLS models developed from FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (RMSE of validation = 8.4% and 11.1% of the range respectively), and IFOP spectra were successfully used to predict elastin content (RMSE = 11.3% of the range). The PLS regression coefficients from the FT-IRIS models were used to map collagen and elastin in tissue sections of degraded porcine aortic tissue as well as a human AAA biopsy tissue, creating a similar map of each component compared to histology. These results support further application of FTIR spectroscopic techniques for evaluation of AAA tissues.

Validation of the modified Vesikari score in children with gastroenteritis in 5 US emergency departments.

OBJECTIVES: The burden of acute gastroenteritis (AGE) in US children is substantial. Research into outpatient treatment strategies has been hampered by the lack of easily used and validated gastroenteritis severity scales relevant to the populations studied. We sought to evaluate, in a US cohort, the reliability, construct validity, and generalizability of a gastroenteritis severity scale previously derived in a Canadian population, the modified Vesikari score (MVS). METHODS: We conducted a prospective, cohort, clinical observational study of children 3 to 48 months of age with acute gastroenteritis presenting to 5 US emergency departments. A baseline MVS score was determined in the emergency department, and telephone follow-up 14 days after presentation was used to assign the follow-up MVS. We determined reliability using inter-item correlations; construct validity via principal component factor analysis; cross-sectional construct validity via correlations with the presence of dehydration, hospitalization, and day care and parental work absenteeism; and generalizability via score distribution among sites. RESULTS: Two hundred eighteen of 274 patients (80%) were successfully contacted for follow-up. Cronbach α was 0.63, indicating expectedly low internal reliability because of the multidimensional properties of the MVS. Factor analysis supported the appropriateness of retaining all variables in the score. Disease severity correlated with dehydration (P < 0.001), hospitalization (P < 0.001), and subsequent day care (P = 0.01) and work (P < 0.001) absenteeism. The MVS was normally distributed, and scores did not differ among sites. CONCLUSIONS: The MVS effectively measures global severity of disease and performs similarly in varying populations within the US health care system. Its characteristics support its use in multisite outpatient clinical trials.

Heme oxygenase-1-mediated autophagy protects against hepatocyte cell death and hepatic injury from infection/sepsis in mice.

UNLABELLED: Adaptive responses to sepsis are necessary to prevent organ failure and death. Cellular signaling responses that limit cell death and structural damage allow a cell to withstand insult from sepsis to prevent irreversible organ dysfunction. One such protective pathway to reduce hepatocellular injury is the up-regulation of heme oxygenase-1 (HO-1) signaling. HO-1 is up-regulated in the liver in response to multiple stressors, including sepsis and lipopolysaccharide (LPS), and has been shown to limit cell death. Another recently recognized rudimentary cellular response to injury is autophagy. The aim of these investigations was to test the hypothesis that HO-1 protects against hepatocyte cell death in experimental sepsis in vivo or LPS in vitro via induction of autophagy. These data demonstrate that both HO-1 and autophagy are up-regulated in the liver after cecal ligation and puncture (CLP) in C57BL/6 mice or in primary mouse hepatocytes after treatment with LPS (100 ng/mL). CLP or LPS results in minimal hepatocyte cell death. Pharmacological inhibition of HO-1 activity using tin protoporphyrin or knockdown of HO-1 prevents the induction of autophagic signaling in these models and results in increased hepatocellular injury, apoptosis, and death. Furthermore, inhibition of autophagy using 3-methyladenine or small interfering RNA specific to VPS34, a class III phosphoinositide 3-kinase that is an upstream regulator of autophagy, resulted in hepatocyte apoptosis in vivo or in vitro. LPS induced phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), in part, via HO-dependent signaling. Moreover, inhibition of p38 MAPK prevented CLP- or LPS-induced autophagy. CONCLUSION: Sepsis or LPS-induced autophagy protects against hepatocellular death, in part via an HO-1 p38 MAPK-dependent signaling. Further investigations are needed to elucidate how autophagic signaling prevents apoptosis and cell death.

Lipopolysaccaride induces autophagic signaling in macrophages via a TLR4, heme oxygenase-1 dependent pathway.

Toll-like receptor (TLR) signaling is an important part of the innate immune response. One of the downstream responses to TLR4 signaling upon lipopolysaccharide (LPS) stimulation is the induction of autophagy, which is a key response to multiple stressors. An additional adaptive signaling molecule that is involved in the response to stress is heme oxygenase-1 (HO-1). HO-1 signaling is essential to limit inflammation and restore homeostasis. We found that LPS induced autophagic signaling in macrophages via a TLR4, HO-1 dependent pathway in macrophages. These data add to the developing contribution of autophagic signaling as part of the inflammatory response.

Heme oxygenase 1 protects against hepatic hypoxia and injury from hemorrhage via regulation of cellular respiration.

Heme oxygenase 1 (HO-1) is an important regulator of the cellular response to stress and inflammation. These investigations test the hypothesis that HO-1 overexpression protects against hemorrhage-induced hypoxia by regulating cellular respiration and oxygen availability. Male C57BL/6 mice or primary mouse hepatocytes were treated with adenoviral gene transfer of HO-1 (AdHO-1) or beta-galactosidase (AdLacZ). Mice were subjected to hemorrhagic shock and resuscitation or cannulation without hemorrhage. AdHO-1 prevented hemorrhagic shock/resuscitation-induced liver injury. In addition, AdHO-1 prevented hemorrhage-induced liver hypoxia and depletion of adenosine triphosphate. In vitro, HO-1 overexpression resulted in decreased cellular respiration under hypoxic conditions as determined by oxygen consumption and cytochrome c oxidase activity. This resulted in increased intracellular oxygen levels in the setting of low oxygen tensions. In conclusion, HO-1 overexpression protects the liver against hemorrhage-induced injury. This may be secondary to the ability of HO-1 to protect against bioenergetic failure via regulation of cellular respiration.

Nitrite potently inhibits hypoxic and inflammatory pulmonary arterial hypertension and smooth muscle proliferation via xanthine oxidoreductase-dependent nitric oxide generation.

BACKGROUND: Pulmonary arterial hypertension is a progressive proliferative vasculopathy of the small pulmonary arteries that is characterized by a primary failure of the endothelial nitric oxide and prostacyclin vasodilator pathways, coupled with dysregulated cellular proliferation. We have recently discovered that the endogenous anion salt nitrite is converted to nitric oxide in the setting of physiological and pathological hypoxia. Considering the fact that nitric oxide exhibits vasoprotective properties, we examined the effects of nitrite on experimental pulmonary arterial hypertension. METHODS AND RESULTS: We exposed mice and rats with hypoxia or monocrotaline-induced pulmonary arterial hypertension to low doses of nebulized nitrite (1.5 mg/min) 1 or 3 times a week. This dose minimally increased plasma and lung nitrite levels yet completely prevented or reversed pulmonary arterial hypertension and pathological right ventricular hypertrophy and failure. In vitro and in vivo studies revealed that nitrite in the lung was metabolized directly to nitric oxide in a process significantly enhanced under hypoxia and found to be dependent on the enzymatic action of xanthine oxidoreductase. Additionally, physiological levels of nitrite inhibited hypoxia-induced proliferation of cultured pulmonary artery smooth muscle cells via the nitric oxide-dependent induction of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The therapeutic effect of nitrite on hypoxia-induced pulmonary hypertension was significantly reduced in the p21-knockout mouse; however, nitrite still reduced pressures and right ventricular pathological remodeling, indicating the existence of p21-independent effects as well. CONCLUSIONS: These studies reveal a potent effect of inhaled nitrite that limits pathological pulmonary arterial hypertrophy and cellular proliferation in the setting of experimental pulmonary arterial hypertension.

Carbon monoxide activates NF-kappaB via ROS generation and Akt pathways to protect against cell death of hepatocytes.

Heme oxygenase overexpression or exogenous carbon monoxide (CO) protects against hepatocyte apoptosis and fulminant hepatitis. The prevention of hepatocyte apoptosis by CO has been shown to require activation of NF-kappaB. The purpose of these investigations was to determine the mechanism of CO-induced hepatocyte NF-kappaB activation and protection against apoptosis. Primary rat or mouse hepatocytes and Hep3B cells were utilized. CO exposure was performed at 250 parts per million. Main outcome measures included cell viability, reactive oxygen species (ROS) generation, and changes in the levels of the intracellular antioxidants glutathione and ascorbate. Western blotting was performed for phospho-Akt, total Akt, and IkappaBalpha. NF-kappaB activation was determined by electrophoretic mobility shift assay and luciferase reporter assays. We found that CO treatment of hepatocytes prevents spontaneous apoptosis and leads to an increase in ROS production in association with Akt phosphorylation and IkappaB degradation. CO did not increase ROS production in respiration-deficient (rho0) Hep3B cells. Both Akt phosphorylation and IkappaB degradation can be inhibited by the addition of antioxidants. Furthermore, CO-induced NF-kappaB activation is reversed by phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY294002) or antioxidants. Additionally, prevention of spontaneous hepatocyte apoptosis by CO is reversed by PI3-K inhibition and antioxidants. In conclusion, these data implicate a survival pathway of CO-induced ROS, Akt phosphorylation, and NF-kappaB activation in cultured hepatocytes. This pathway may prove to be important in maintenance of hepatic function in both physiological and pathophysiological conditions.

Prolonged exposure to reduced levels of androgen accelerates prostate cancer progression in Nkx3.1; Pten mutant mice.

In this report, we have investigated the relationship between androgen levels and prostate tumorigenesis in Nkx3.1; Pten mutant mice, a genetically engineered mouse model of human prostate cancer. By experimentally manipulating serum levels of testosterone in these mice for an extended period (i.e., 7 months), we have found that prolonged exposure of Nkx3.1; Pten mutant mice to androgen levels that are 10-fold lower than normal (the "Low-T" group) resulted in a marked acceleration of prostate tumorigenesis compared with those exposed to androgen levels within the reference range (the "Normal-T" group). We found that prostate tumors from the Low-T mutant mice share a similar gene expression profile as androgen-independent prostate tumors from these mutant mice, which includes the deregulated expression of several genes that are up-regulated in human hormone-refractory prostate cancer, such as Vav3 and Runx1. We propose that exposure to reduced androgens may promote prostate tumorigenesis by selecting for molecular events that promote more aggressive, hormone-refractory tumors.

Carbon monoxide signals via inhibition of cytochrome c oxidase and generation of mitochondrial reactive oxygen species.

Carbon monoxide (CO), which is produced endogenously in the breakdown of heme, has been recognized as an important physiological second messenger similar to NO. Additionally, pharmacological delivery of CO is protective in numerous models of injury, including ischemia/reperfusion, transplantation, hemorrhagic shock, and endotoxemia. However, the mechanism of action of CO is only partially elucidated focused primarily on how it modulates the cellular response to stress. The purpose of these investigations is to test the hypothesis that CO acts via inhibition of cytochrome c oxidase leading to the generation of low levels of reactive oxygen species (ROS) that in turn mediate subsequent adaptive signaling. We show here that CO increases ROS generation in RAW 264.7 cells, which is inhibited by antimycin A and is absent in respiration-deficient rho0 cells. CO inhibits cytochrome c oxidase, while maintaining cellular ATP levels and increasing mitochondrial membrane potential. The addition of antioxidants or inhibition of complex III of the electron transport chain by antimycin A attenuates the inhibitory effects of CO on lipopolysaccharide (LPS)-induced TNF-alpha and blocked CO-induced p38 MAPK phosphorylation, which we previously have shown to be important in the anti-inflammatory effects of CO.

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA.

Nitric oxide (NO) acts as a vasoregulatory molecule that inhibits vascular smooth muscle cell (SMC) proliferation. Studies have illustrated that NO inhibits SMC proliferation via the extracellular signal-regulated kinase (ERK) pathway, leading to increased protein levels of the cyclin-dependent kinase inhibitor p21(Waf1/Cip1). The ERK pathway can be pro- or antiproliferative, and it has been demonstrated that the activation status of the small GTPase RhoA determines the proliferative fate of ERK signaling, whereby inactivation of RhoA influences ERK signaling to increase p21(Waf1/Cip1) and inhibit proliferation. The purpose of these investigations was to examine the effect of NO on RhoA activation/S-nitrosation and to test the hypothesis that inhibition of SMC proliferation by NO is dependent on inactivation of RhoA. NO decreases activation of RhoA, as demonstrated by RhoA GTP-binding assays, affinity precipitation, and phalloidin staining of the actin cytoskeleton. Additionally, these effects are independent of cGMP. NO decreases SMC proliferation, and gene transfer of constitutively active RhoA (RhoA(63L)) diminished the antiproliferative effects of NO, as determined by thymidine incorporation. Western blots of p21(Waf1/Cip1) correlated with changes in proliferation. S-nitrosation of recombinant RhoA protein and immunoprecipitated RhoA was demonstrated by Western blotting for nitrosocysteine and by measurement of NO release. Furthermore, NO decreases GTP loading of recombinant RhoA protein. These findings indicate that inactivation of RhoA plays a role in NO-mediated SMC antiproliferation and that S-nitrosation is associated with decreased GTP binding of RhoA. Nitrosation of RhoA and other proteins likely contributes to cGMP-independent effects of NO.

Carbon monoxide reverses established pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by a progressive increase in pulmonary vascular resistance leading to right heart failure. Carbon monoxide (CO) has emerged as a potently protective, homeostatic molecule that prevents the development of vascular disorders when administered prophylactically. The data presented in this paper demonstrate that CO can also act as a therapeutic (i.e., where exposure to CO is initiated after pathology is established). In three rodent models of PAH, a 1 hour/day exposure to CO reverses established PAH and right ventricular hypertrophy, restoring right ventricular and pulmonary arterial pressures, as well as the pulmonary vascular architecture, to near normal. The ability of CO to reverse PAH requires functional endothelial nitric oxide synthase (eNOS/NOS3) and NO generation, as indicated by the inability of CO to reverse chronic hypoxia-induced PAH in eNOS-deficient (nos3-/-) mice versus wild-type mice. The restorative function of CO was associated with a simultaneous increase in apoptosis and decrease in cellular proliferation of vascular smooth muscle cells, which was regulated in part by the endothelial cells in the hypertrophied vessels. In conclusion, these data demonstrate that CO reverses established PAH dependent on NO generation supporting the use of CO clinically to treat pulmonary hypertension.

Metformin adjunctive therapy with insulin improves glycemic control in patients with type 1 diabetes mellitus: a pilot study.

Metformin lowers blood glucose by reducing hepatic glucose output and improving insulin sensitivity without requiring an increase in circulating insulin concentration. We hypothesized that metformin could be used adjunctively with insulin to improve glycemic control in type 1 diabetes mellitus (DM). We conducted a 6-month open-label pilot study in 10 adolescents and young adults with type 1 DM, 19.1 +/- 3.4 years, 4 males, 6 females, and body mass index 26.3 +/- 3.1 kg/m2. Patients started metformin at a dose of 250 mg b.i.d.; the dose was increased until blood glucose was within an optimal target range or a maximum of 2,500 mg/d was reached. Insulin dose was reduced as needed to prevent hypoglycemia. Seven patients had an average decrease in HbA(1c) of 11% from pretreatment. These responders had no change in insulin dose, BMI or lipid levels during the study. Three patients had no improvement of HbA(1c) on therapy. We conclude that some patients with type 1 DM will have improved glycemic control on adjunctive metformin therapy. Evaluation of the long-term benefit and safety of adjunctive therapy in patients with type 1 DM is warranted.